The continuing overall goal of our research program is to understand the relationship between functional MRI (fMRI) signals and the underlying neural activity. During the previous period of support, we focused on elucidating specific neurovascular, neuroglial, and neurometabolic events that affect positive or negative hemodynamic response. We improved the methodology for quantitative imaging of calcium, validated a new technology for microscopic imaging of partial pressure of O2 (pO2), and took aboard optogenetics for manipulation of specific cell types that became available within the duration of the support period. With these methods in hand, we probed dilation/constriction of cerebral microvasculature following neuroglial calcium- dependent release of vascular mediators, intravascular and tissue pO2, and layer-resolved BOLD fMRI responses as a function of the underlying vascular dynamics. The key implication of our study is that cerebral blood flow (CBF) and cerebral metabolic rate of O2 (CMRO2) are being driven in parallel by neural activity, and potentially by different aspects of neural activity. For example, activation o the excitatory cells is associated with large metabolic costs of repolarization, glutamate recycling and calcium buffering but produces smaller dilation compared to activation of the inhibitory neurons. In parallel, similar conclusions have been reached by our co-investigator Dr. Buxton based on human studies that utilized the calibrated BOLD fMRI technique. Taken together with the insights from our microscopic imaging and manipulation, these results lead us to put forward the hypothesis that variability in the ratio of the fractional changes in CBF and CMRO2 reflects different proportions of inhibitory and excitatory evoked activity. If proven true, this would open a new direction in which quantitative fMRI may be able to provide information on the underlying neural activity. Thus, the goal of this proposal is to investigate the behavior o the CBF/CMRO2 ratio using microscopic imaging of the relevant underlying physiological parameters while directly controlling neural activity. Aim 1 is focused on exploring the CBF/CMRO2 ratio during selective optogenetic activation of excitation and inhibition in the mouse sensory cortex in vivo. In Aim 2, we will use optogenetics for modulating the baseline CBF and CMRO2 mimicking the neural response to a standard stimulus shaped by different brain states. The proposed project is an integral part our collaborative research program where animal and human efforts progress in a continual dialog. While the CBF/CMRO2 ratio hypothesis has been derived in part from human studies, the human data provide no direct information on the balance of excitatory and inhibitory activity. Our set of imaging and manipulation tools in the mouse, on the other hand, is perfectly suited to evaluate this hypothesis under well-controlled conditions. The proposed animal experiments will lay a mechanistic foundation for clinical applications of the calibrated BOLD methodology potentially enabling a paradigm shift in human fMRI studies: from simply asking where activation occurs to asking how much activation occurs.